1. Field of the Invention
This invention relates to a new anticoagulant for human use and to a process for producing and a method for using the new anticoagulant.
Heparin ("Hep") is a known blood anticoagulant that functions by binding tthe inhibitor, antithrombin (anti thrombin-III or "AT-III"), and by accelerating the rate at which this inhibitor neutralizes serum proteases of the coagulation system. Heparin has been fractionated into components having varying degrees of activity and there is evidence which suggests that only a very small proportion of unfractionated heparin constitutes that component which is characterized by relatively high specific activity.
Although unfractionated heparin alone is routinely employed for the treatment of thromboembolism or for the prevention of clot formation in at-risk patients, problems concerning side effects and efficacy have been pointed out. By the expression "at-risk patients" is meant those patients prone to develop thromboembolisms. For example, Rao et al, Thromb. Res., 24, 181 (1981) and Boneu et al, Thromb. Res., 27, 123 (1982) have noted that continuous intravenous infusion of heparin results in a decrease (about 35%) in plasma antithrombin-III ("AT-III") levels in all thromboembolic patients receiving heparin therapy. These patients may be at an enhanced risk. Baird et al, J. Bone Jt. Surg., 59A, 1061 (1977), report recurrent arterial thrombosis in patients on systemic heparin therapy.
Further, although the administration of antithrombin has been proposed as a means at controlling undesirable clot-formation in at-risk patients, especially for those patients having congenital antithrombin deficiency, this therapy would require very large amounts of antithrombin and frequent administration of antithrombin since it has a plasma half-life of about three days.
Antithrombin - heparin complexes have been reported and proposed for therapeutic use in anticoagulant therapy. Further, preparation of such a complex in pharmaceutically useful amounts has been reported.
2. Related Application
U.S. Ser. No. 357,504, filed Mar. 12, 1982, a continuation-in-part of U.S. Ser. No. 192,170, filed Sept. 30, 1980 and now abandoned, both being owned by the assignee of the present application, discloses an antithrombin-high activity heparin complex produced by contacting a lectin-containing, water-insoluble matrix with antithrombin and heparin to allow (1) the antithrombin tbind reversibly with the matrix and (2) the high activity heparin component of heparin to complex with the antithrombin, washing the matrix tremove unbound heparin, contacting the matrix with a solution of a carbohydrate having the ability tdisplace the antithrombin-high activity heparin complex, and recovering the complex free of uncomplexed heparin and antithrombin.
3. Description of the Prior Art
Rosenberg et al, Proc. Natl. Acad. Sci., 75 (7), 3065 (1978), disclose the fractionation of porcine heparin species of low molecular weight into highly active and relatively inactive fractions by a 2-cycle affinity technique based on the affinity of heparin for antithrombin. There were obtained three heparin fractions, namely (1) a "highly active heparin" from Cycle I of the process, and (2) an "active" and (3) a "relatively inactive" heparin species both from Cycle II of the process. "Active" and "highly active" heparin species were each obtained as a complex with antithrombin after separation from unbound heparin by gel filtration on Sephadex G-100. The "highly active heparin"-antithrombin complex was obtained in Cycle I by chromatography of a mixture of antithrombin and low molecular weight heparin at a molar ratiof 0.08/1.0. The "active heparin"-antithrombin complex was obtained in Cycle II by similarly chromatographing a mixture of antithrombin and the residual unbound heparin from Cycle I at a molar ration of 1.5/1.0. Both of these heparin fractions were separated from antithrombin in separate, subsequent chromatography steps on G-100 in the presence of 3 M NaCl. "Relatively inactive" heparin was obtained in Cycle II as that fraction of heparin which did not bind to antithrombin. The resulting highly active, active and relatively inactive heparin species were subjected to analysis tdetermine their relative abundance, specific anticoagulant activity, composition and structure.
Rosenberg et al, Biochem. Biophys. Res. Commun., 86 (4), 1319 (1979), disclose the results of a study of the anticoagulant activity of porcine heparin fractions obtained by gel filtration and affinity fractionation and provide the first demonstration that heparin molecules may bear multiple binding sites for antithrombin.
Rosenberg, U.S. Pat. No. 4,301,153, discloses a heparin preparation which exhibits elevated anticoagulant activity and a process for producing the preparation. Conventional, heterogenous heparin is incubated with antithrombin-III, the "heparin cofactor", and a portion of the heparin forms a complex with antithrombin-III. The uncomplexed and complexed heparin fractions are separated, and the complexed fraction is broken down to obtain antithrombin-III and an "active" form of heparin.
Jordan et al, J. Biol. Chem., 254 (8), 2902 (1979), disclose the preparation of low molecular weight porcine heparin having average specific anticoagulant activity of 94 units/mg and affinity fractionation thereof, using techniques described in the Rosenberg references mentioned above, intforms having varying degrees of activity with respect tthe affinity of heparin for binding with antithrombin. Further, the reference discloses the results of the examination of the ability of the active heparin fraction, especially the highly active heparin fraction, to accelerate the thrombin - antithrombin interaction which demonstrated that heparin functions as a catalyst in the thrombin - antithrombin interaction.
Lam et al, Biochem, Biophys. Res. Commun., 69 (2), 570 (1976), disclose the fractionation of heparin intactive and inactive forms by sucrose density gradient centrifugation of heparin mixed with antithrombin - thrombin cofactor and the first demonstration that only a small fraction of a given heparin preparation can bind to antithrombin - thrombin cofactor and is responsible for its distinctive anticoagulant effect.
Hook et al, FEBS Letters, 66 (1), 90 (1976), disclose the separation of high-activity heparin and low-activity heparin by affinity chromatography on an antithrombin substituted affinity matrix. For example, bovine anti-thrombin was coupled via amingroups tcyanogen bromide activated Sepharose.RTM. 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) in the presence of excess heparin (radioactive heparin previously treated with acetic anhydride in order to acetylate any free amingroups) added tshield the heparin binding sites of the antithrombin molecule from binding tthe Sepharose beads. Affinity chromatography of heparin on the immobilized antithrombin was carried out by applying samples of heparin, in 1 ml of 0.2 M NaCl-0.1 M Tris HCl, pH 7.4, to a column containing 3 ml of antithrombin--Sepharose gel equilibrated with the same buffer at 4.degree. C. After washing with buffer until the effluent was free of uronic acid and radioactivity, the adsorbed heparin was eluted with a linear salt gradient, that is, buffered 3 M NaCl. Two distinct fractions were obtained. A portion of the material, low-affinity heparin, was obtained in the break-through fraction (or effluent) and another portion, high-affinity heparin, was obtained during gradient elution.
All of the foregoing antithrombin - heparin complexes, however, are ionic association complexes (not covalently linked) and upon infusion into small animals have not shown significant prolongation of anticoagulant properties compared to heparin controls. Dissociation of the ionic complex and subsequent inactivation of the components by ordinary biological means immediately following infusion is a likely explanation of this observation.
If dissociation of the ionic complex has been the major factor resulting in no significant prolongation of anticoagulant properties of the ionic complexes, then covalent attachment between antithrombin-III and heparin was conceived as a solution to this problem. According to this conception, heparin, a sulfonated linear mucopolysaccharide with a mean molecular weight range of 7,600-19,700 (polymeric size range of 5,000-30,000 daltons), has hydroxyl groups which could be expected to be available for activation, in liquid phase, by cyanogen halides in basic pH conditions of about 10.7-11.0. The resulting imidocarbonate could then be coupled to purified antithrombin-III at about pH 9.0 and the residual active groups neutralized with glycine. Support, by analogy, for this concept is found in Tam et al, Proc. Natl. Acad. Sci., 73 (6), 2128 (1976) who demonstrated that, using dextran as the carbohydrate, a cyanogen bromide coupled dextranhemoglobin complex was cleared from circulation much more slowly than was free hemoglobin. Also, Marshall et al, J. Biol. Chem., 254 (4), 1081 (1976) similarly formed a covalent dextran--trypsin conjugate using cyanogen bromide and demonstrated that intramolecular crosslinking of the protein by polysaccharide results in stabilization of the tertiary structure. Further, Kohn et al, Enzyme Microb. Technol. 4, 161 (1982) and Biochem. Biophys. Res. Commun., 107 (3), 878 (1982), describe the results of a study of the mechanism of CNBr activation of polysaccharide resins like Sepharose and Sephadex and a new approach for the chemical reaction of Sepharose with CNBr, respectively.
Covalent complexes between heparin and antithrombin have been reported. Hoylaerts et al, Thromb. Haemostas., (Stuttgart), 49 (2), 109 (1983), and Ceustermans et al, J. Biol. Chem., 257 (7), 3401 (1982), disclose the chemical coupling of high affinity heparin fragments of low molecular weight heparin and of intact high affinity heparin to antithrombin-III to yield covalent complexes which inhibited factor Xa with a second order rate constant similar to those obtained for antithrombin-III saturated with heparin fragments and to that obtained for the covalent complex between antithrombin-III and native high affinity heparin. Primary amino groups, which were introduced in the high affinity fraction by N-sulfation followed by substitution with hexamethylenediamine, were reacted under basic conditions of pH with tolylene-2,4-diisothiocyanate and the isothiocyanate-containing intermediate was extracted and reacted in buffer solution at basic pH and at 30.degree. C. with antithrombin. The resulting mixture of antithrombin-III, heparin and their covalent complex was separated by affinity chromatographic and ion exchange techniques.
Bjork et al, FEBS Letters, 143 (1), 96 (1982), disclose the permanent activation of antithrombin by covalent attachment to high affinity heparin oligosaccharides, which oligosaccharides were obtained by treatment of heparin with nitrous acid thus leading to formation of fragments having 2,5-D-anhydromannose residues with reactive aldehyde functions at their reducing terminals. The oligosaccharides thus obtained were covalently attached to antithrombin by a procedure based on the formation of a labile Schiff's base between the anhydromannose residue of the oligosaccharide and the amino group of a neighboring lysine residue of the protein.